This invention relates to a method and microfabricated device for sorting cells or particles by size, charge or other identifying characteristics, for example, characteristics that can be optically detected. The invention includes a fluorescence activated cell sorter (FACS), and methods for analyzing and sorting cells by measuring a signal produced by an optically-detectable (e.g., fluorescent, ultraviolet or color change) reporter associated with the cells. The methods and apparatus of the invention allow for high sensitivity, no cross-contamination, and lower cost than conventional FACS machines. In preferred embodiments, cell sorting is performed on a microfabricated chip with a detection volume of approximately 1 to 1,000,000 femtoliters (fl), preferably about 200 to 500 fl, and most preferably about 375 fl. Sorting occurs immediately after detection. In a particular embodiment, the inlet and collection wells are incorporated on the same chip.
Sorters of the invention can function as stand-alone devices or as components of integrated microanalytical chips, and can be disposable. Living cells with a distinguishing characteristic, such as E. coli cells expressing a fluorescent protein, can be efficiently separated from cells lacking this characteristic. Furthermore, the cells remain viable after being extracted from the sorting device. An advantage of the invention is that it can be applied to various aspects of chemical and biological studies, e.g., cell sorting, enzyme catalysis and molecular evolution (1).
The references cited herein are referred to numerically, and are appended in a Bibliography below. All of the references are incorporated herein in their entirety.
Harrison et al. (39) disclose a microfluidic device which manipulates and stops the flow of fluid through a microfabricated chip, so that a cell can be observed after it interacts with a chemical agent. The cells and the chemical agent are loaded into the device via two different inlet channels which intersect with a main flow path. The flow of the fluid is controlled by a pressure pump or by electric fields (electrophoretic or electro-osmotic) and can be stopped so that the cells can be observed, after they mix and interact with the chemical. The cells then pass through the main flow path, which terminates in a single common waste chamber. Harrison et al. do not provide a device or method for sorting cells, nor do they suggest or motivate one having ordinary skill in the art to make and use any such device. On the contrary, cells are mixed with chemicals, observed, and are discarded as waste.
Conventional flow cell sorters, such as FACS, are designed to have a flow chamber with a nozzle and use the principle of hydrodynamic focusing with sheath flow to separate or sort biological material such as cells (2-7). In addition, most sorting instruments combine the technology of ink-jet writing and the effect of gravity to achieve a high sorting rate of droplet generation and electrical charging (8-10). Despite these advances, many failures of these instruments are due to problems in the flow chamber. For example, orifice clogging, particle adsorption and contamination in the tubing may cause turbulent flow in the jet stream. These problems contribute to the great variation in illumination and detection in conventional FACS devices. Another major problem is known as sample carryover, which occurs when remnants of previous specimens left in the channel back-flush into the new sample stream during consecutive runs. A potentially more serious problem occurs when dyes remain on the tubing and the chamber, which may give false signals to the fluorescence detection or light scattering apparatus. Although such systems can be sterilized between runs, it is costly, time consuming, inefficient, and results in hours of machine down time for bleaching and sterilization procedures.
Similarly, each cell, as it passes through the orifice, may generate a different perturbation in response to droplet formation. Larger cells can possibly change the droplet size, non-spherical cells tend to align with the long axis parallel to the flow axis, and deformable cells may elongate in the direction of the flow (9, 10). This can result in some variation in the time from the analysis to the actual sorting event. Furthermore, a number of technical problems make it difficult to generate identically charged droplets, which increases deflection error. A charged droplet may cause the next droplet of the opposite polarity to have a reduced charge. On the other hand, if consecutive droplets are charged identically, then the first droplet might have a lower potential than the second droplets, and so on. Yet, charged droplets will have a defined trajectory only if they are charged identically. In addition, increasing droplet charges may cause mutual electrostatic repulsion between adjacent droplets, which also increases deflection error. Other factors, such as the very high cost for even modest conventional FACS equipment (on the order of $250,000), the high cost of maintenance, and the requirement for trained personnel to operate and maintain the equipment have been among the main considerations that hinder this technology and its widespread accessibility and use (10). Even though the field of flow cytometry has been extensively exploited in the development of cell sorting devices, significant problems persist and remain to be addressed. Thus, there is a need for improved methods and machines for cell sorting which are fast, efficient, costeffective and disposable.
The invention provides a microfabricated device for sorting cells based on a desired characteristic, for example, reporter-labeled cells can be sorted by the presence or level of reporter on the cells. The device includes a chip having a substrate into which is microfabricated at least one analysis unit. Each analysis unit includes a main channel, having a sample inlet channel, typically at one end, and a detection region along its length. Adjacent and downstream from the detection region, the main channel has a discrimination region or branch point leading to at least two branch channels. The analysis unit may further include additional inlet channels, detection points, branch points, and branch channels as desired. A stream containing the cells, e.g., in a solution or mixture, is passed through the detection region, such that on average only one cell occupies the detection region at any given time. The cells can be sorted based on their ability to emit a detectable signal such as an optical signal, with or without stimulation, such as exposure to light in order to promote fluorescence. According to the invention, the presence or level of reporter from each cell is measured within the detection region, and each cell is directed to a selected branch channel based on the level of reporter detected or measured.
In addition to sorting fluorescent and non-fluorescent cells, the invention can also provide multiparameter analysis, such as multicolor detection or a gated window detection. For example, beads of different colors, or cells labelled with one or more chromophores, can be sorted by the invention. Sorting according to a window, or threshold, means that cells or particles are selected for sorting based on the presence of a signal above a certain value or threshold, and which is typically lower than a certain upper limit. There can also be several points of analysis on the same chip for multiple time course measurements.
The invention offers several advantages over traditional sheath flow methods. Since the channels in the present device can be made with micron dimensions, the volume of the detection region is precisely controlled and there is no need for hydrodynamic focusing. The planar geometry of the device allows the use of high numerical aperture optics, thereby increasing the sensitivity of the system. Since fluid flows continuously through the system, there is no need for droplet formation, or for charged droplets, and many challenging technical issues can be avoided. In addition, there is no aerosol formation because the system is entirely self-contained, allowing much safer sorting of biohazardous material, in comparison with conventional FACS devices. The sorting device of the invention is also disposable, which obviates the need for cleaning and sterilizing the instrument, and prevents cross-contamination between samples.
Thus, a cell sorter of the invention, such as a disposable microfabricated FACS, employs a substrate that integrates at least one inlet channel and at least two outlet channels, which meet at a branch or sorting point. In a preferred embodiment, the substrate is planar, and contains a microfluidic chip made from a silicone elastomer impression of an etched silicon wafer according replica methods in soft-lithography (11). In one embodiment, the channels meet to form a xe2x80x9cTxe2x80x9d (T junction). A Y-shaped junction, and other shapes and geometries may also be used. A detection region is typically upstream from the branch point. Cells are diverted into one or another outlet channel based on a predetermined characteristic that is evaluated as each cell passes through the detection region. The channels are preferably sealed to contain the flow, for example by fixing a transparent coverslip, such as glass, over the chip, to cover the channels while permitting optical examination of one or more channels or regions, particularly the detection region. In a preferred embodiment the coverslip is pyrex, anodically bonded to the chip.
In one embodiment, cells are directed into one or another of a pair of outlet channels by electrodes that apply an electric field across the branch point, which effectively directs a particular cell into a predetermined outlet or branch channel.
In another embodiment, a flow of cells is maintained through the device via a pump or pressure differential. A valve structure at the branch point permits each cell to enter only one of the branch channels depending on the measurement at the detection point. In a similar embodiment, a valve structure can be provided for each branch channel, downstream of the branch point, which allows or curtails the flow through a particular channel. Alternatively, the pressure may be adjusted within or at the outlet of each branch channel, to allow or curtail flow through the channel.
An apparatus, machine or device of the invention may include a plurality of analysis units, and in such embodiments can further include a plurality of manifolds (e.g., a fitting or point with more than one lateral outlet to permit connection of or division to branch channels). The number of manifolds typically equals the number of branch channels in one analysis unit, to facilitate collection of cells from corresponding branch channels of the different analysis units.
The microfabricated device includes a transparent coverslip (e.g., glass) bonded to the substrate and covering the channels to form a xe2x80x9croofxe2x80x9d and/or xe2x80x9cfloorxe2x80x9d for the channels. A silicon chip with an anodically bonded pyrex coverslip may be used. The channels in the device are preferably between about 1 and 500 microns in width and between about 1 and 500 microns in depth, and the detection region has a volume of between about 1 fl and 100 nl.
Where desired, an external laser, a diode or integrated semiconductor laser or a high-intensity lamp (e.g., a mercury lamp) may be used to stimulate a reporter to release a measurable or detectable signal (e.g., light energy). Measurements may be taken, for example, using a microscope in connection with an intensified charge couple device (CCD) camera, photomultiplier tube, avalanche photodiode, an integrated photodiode, or the like.
In another aspect, the invention includes a method of isolating cells having a selected threshold amount of a bound or associated optically-detectable (e.g., fluorescent, ultraviolet or color change) reporter. The method includes, (a) flowing a stream of solution containing reporter-labeled cells through a channel comprising a detection region having a selected volume, where the concentration of the cells in the solution is such that they pass through the detection region one by one, (b) determining the presence or amount of reporter on each cell as it passes through the detection region, (c) diverting cells having a selected threshold of reporter into a first branch channel, and diverting cells not having the selected threshold into a second branch channel, and (d) collecting cells diverted into one or more branch channels.
The method can be applied to diverting a cell having a selected reporter threshold into the first branch channel, in such a way that the diverting action blocks the flow into the second branch channel. That is, the second channel is blocked and the stream carries the cell having the selected reporter threshold into the first branch channel. Alternatively or in addition, the method may be used to divert a cell that does not have the selected reporter threshold into the second branch channel, by blocking the flow into the first branch channel. This can be done, for example, using a valve or valves that are actuated by an electrical or mechanical switch responsive to a reporter measurement.
The method may be applied to any cell, including prokaryotic or eukaryotic, such as bacterial, plant, animal, and the like. The method is particularly useful for the sorting of mammalian (e.g., human) blood cells, such as peripheral blood mononuclear cells (PBMCs), based on the expression of various antigens, such as HLA DR, CD3, CD4, CD8, CD11a, CD11c, CD14, CD16, CD20, CD45, CD45RA, CD62L, etc. The method can also be used to sort any cell on the basis of whether it does or does not express or produce a detectable protein, either directly or in cooperation with a reporter molecule. For example, cells that produce a fluorescent protein may be sorted from those that do not. Alternatively, a fluorescent protein can be used as a reporter, for example, by co-expression with another protein (50, 51).
Alternatively, the cell may produce a detectable substance (e.g. a fluorescent compound) through its interaction with another substance added to the fluid medium. For example, cells containing a gene for a monooxygenase enzyme may catalyze a reaction on an aromatic substrate (e.g. benzene or naphthalene) with the net result that the fluorescence, or another detectable property of the substrate, will change. This change can be detected in the detection region, and cells having that change in fluorescence can be collected based on predetermined criteria. A second reagent or coupling enzyme can be used to enhance fluorescence. See, Affholter and Arnold (50) and Joo et al. (51).